The present invention, in some embodiments thereof, relates to nucleic acid constructs encoding adenosine phosphate-isopentenyltransferase (IPT) under a stress-related promoter and more particularly, but not exclusively, to methods of using same for generating transgenic plants with increased abiotic stress tolerance and increased yield, biomass and growth rate under normal or stress conditions.
Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% and causing losses worth hundreds of million dollars each year. Abiotic stresses lead to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity.
Phenotypic symptoms in response to chilling include induced injury such as leaf expansion, wilting, chlorosis, and necrosis. Chilling also severely hampers the reproductive development of plants and plants may suffer from metabolic dysfunction when chilled.
Freezing conditions cause severe membrane damage, and reactive oxygen species (ROS) produced in response to freeze stress further contributes to membrane damage.
Heat Stress disturbs the cellular homeostasis and can lead to severe retardation in growth and development, and even death.
High salinity, in particular sodium ions (Na+), can dissipate the membrane potential, is toxic to cell metabolism, has deleterious effects on the functioning of some of the plant's enzymes. In addition, high concentrations of Na+ cause osmotic imbalance, membrane disorganization, reduction in growth, inhibition of cell division/expansion, can lead to reduction in photosynthesis and production of reactive oxygen species.
Harsh drought conditions disrupt the normal bilayer structure of the membrane. In addition to membrane damage, cytosolic and organelle protein may exhibit reduced activity or may even undergo complete denaturation when dehydrated. Drought may also cause disruption of cellular metabolism and reduction in vegetative growth, in particular shoot growth.
Abscisic Acid (ABA) plays a primary regulatory role in the initiation and maintenance of seed and bud dormancy and in the plant's response to stress (e.g., freezing, salt stress and water stress (deficit of water). In addition, ABA influences many other aspects of plant development by interacting, usually as an antagonist with auxin, cytokinin, gibberellin, ethylene and brassinosteroids.
Studies of the promoters of several stress-induced genes have lead to the identification of specific regulatory sequences of genes involved in different stresses. A conserved sequence in many ABA-responsive genes has been reported to function as an ABA-responsive element (ABAR), which probably binds to transcription factors involved in ABA-regulated gene activation. A second sequence element found in the promoters of these genes is a dehydration response element (DRE) that is involved in the first rapid response to dehydration or salt. Over-expression of a DRE cDNA in transgenic plants was shown to activate the expression of many stress tolerance genes under normal growing conditions and to improve tolerance to drought, salt loading and freezing.
Deficiencies in macronutrients result in stunted growth, reduced biomass production and hastened senescence of older leaves. Suppression in plant growth and reduced biomass production may be attributed to decreased photosynthetic activity. Plants receiving deficient supplies of sulfur (S) and calcium (Ca) show greater suppression in the growth than nitrogen (N)-deficient plants, probably due to fairly immobile nature of S and Ca. Phosphor (P) deficiency in tobacco plants has been shown to decrease sink demands by limiting growth processes. The changes in the sink demands due to mineral nutrient deficiency may influence the partitioning of photosynthates and dry matter distribution leading to decreased shoot/root ratio.
Nutrient deficiency is predicted to be the single most important factor limiting crop yields during the 21st century, especially in developing countries. Soil acidity, alkalinity and salinity, anthropogenic activities, monoculture farming and wind and water erosion processes are the major degradation factors for cultivated soils. The poor productivity of crops grown in acid and salt affected soils is mainly due to combinations of elemental toxicities and deficiencies or unavailability of essential nutrients. Addition of fertilizers and amendments (particularly lime) are essential in achieving proper nutrient supply and maximizing yields in these soils. However, efficiency of applied fertilizers is very low and varies with crop species and genotype/cultivar within species, and their interactions with the environment. The world's total demand for food is likely to nearly double its present level by 2030, and there is limited new land available for expansion of cultivation to achieve this production level. Therefore, increasing crop yield potentials per unit of land is an urgent concern. The higher nutrient use efficiency in plants must be fully explored to increase food production to feed the growing human population, and this has to be achieved without accelerating environmental degradation from excessive fertilizer use.
Plant senescence, particularly in monocarpic (mostly annuals) species, is a correlatively controlled developmental process encountering at all stages in the life history of plants. However, certain stresses (such as drought and nutrient deficiency) and hormones are able to hasten or repress senescence. During leaf senescence, nutrients are recycled to other parts of the plant such as young leaves or storage tissues. Thus, senescence has a negative impact on yield due to the deterioration of leaf photosynthetic assimilation.
The main players among the plant hormones in the regulation of senescence processes are cytokinin, as the senescence retardant hormone, and ethylene, as the promoting hormone. Cytokinins control various processes in plant growth and development, such as proliferation (promote cell division) and differentiation (e.g., vascular development, leaf expansion, accumulation of chlorophyll and conversion of etioplasts into chloroplasts) of plant cells; they play a role in apical dominance, transduction of nutritional signals, control of shoot-root balance, crop productivity and senescence (delay leaf senescence). Cytokinins are present in all plant tissues and are abundant in root tips, shoot apex and immature seeds. Several studies have shown that a decrease in the flux of cytokinins from the roots up through the xylem is an important factor in the senescence of leaves. The expression level of cytokinin was shown to be highest in new developed tissues and to decrease in mature tissues, enabling senescence initiation.
The biosynthesis of cytokinins is catalyzed by adenosine phosphate-isopentenyltransferase (IPT), and it was found that over-expression of Arabidopsis thaliana IPT (AtIPT) genes results in phenotypes indicative of cytokinin overproduction as they show developmental and morphological alterations (Miyawaki K. et al. 2004, Plant J. 37: 128-38).
Leaf senescence program is accompanied and driven by changes in gene expression. Differential screening of cDNA libraries during senescence demonstrated that the expression of the vast majority of genes is down-regulated, whereas the expression of other genes [senescence-associated genes (SAGs)] is up-regulated (Buchanan-Wollaston V., 1994. Plant Physiol. 105: 839-846; Davies K M and Grieson D., 1989. Planta, 179: 73-80; Lohman K N, et al., 1994. Physiologia Plantarum 92: 322-328; Hajouj T, et al., 2000. Plant Physiol. 124: 1305-1314; Gepstein, S., et al., 2003. The plant journal 36: 629-642; Buchanan-Wollaston V, et al., 2003. Plant Biotechnology Journal 1: 3-22). Among the prominent SAGs are those predicted to be involved in the massive degradation of macromolecules and enzymes for nutrient recycling. Several of the genes which are up-regulated during leaf senescence are also upregulated under abiotic and biotic stresses (Binyamin L, et al., 2000. Planta, 211: 591-597; Buchanan-Wollaston V, 1997. J. Exp. Bot. 48: 181-199; Gepstein et al., 2003 (Supra); Guo Y, et al., 2004. Plant, Cell and Environment 27: 521-549; Hanfrey C, et al., 1996. Plant Mol. Biol. 30: 597-609; Quirino B F, et al., 1999. Plant Mol. Biol. 40: 267-278; Weaver L M, et al., 1997. Leaf senescence: gene expression and regulation. In: Setlow J K, ed. Genetic engineering, Vol 19. New York: Plenum Press, 215-234).
The Senescence-Associated Receptor Kinase (SARK) gene was identified in bean leaves (Phaseolus vulgaris) as an early SAG. The initiation of SARK expression occurs at late stages of leaf maturation, but appears immediately prior to some symptoms of senescence.
Gan S. and Amasino R M., 1995 (Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270: 1986-1988) developed a senescence-inhibition system in which cytokinin production is specifically targeted to the senescence process by transforming plants with a chimeric construct containing the promoter of the highly regulated senescence specific gene-12 (SAG12) fused to the IPT coding sequence (PSAG12-IPT). PSAG12-IPT transgenic plants grew normally until the senescence stage, however, while leaf senescence progressed in the wild-type plants, the transgenic plants showed no visible sign of senescence at this stage.
PCT publication No. WO 2006/102559 discloses the generation of transgenic tobacco plant transformed with a construct containing the SARK promoter fused to the IPT gene. SARK-IPT transgenic plants display significant delay in senescence and enhanced tolerance to drought conditions and minimal reduction in biomass and seed yield of the plants when grown under limited water regime, demonstrating extreme resistance to drought conditions by enhanced photosynthetic rates and water use efficiency, provided by cytokinin expression (Rivero R M, et al., 2007. PNAS 104: 19631-19636). In addition, the cytokinin production in these plants resulted in protection of biochemical processes associated with photosynthesis and in induction of photorespiration, which may contribute to the protection of photosynthesis during water stress (Rivero R M, et al., 2009. Plant Physiol. 150: 1530-40).
Metallothionein (MT) genes encode a family of cysteine-rich, low molecular weight proteins present in a variety of organisms including bacteria, fungi and all eukaryotic plant and animal species which bind heavy metals through the thiol group of their cysteine (Cys) residues. Metallothioneins were found to be induced in several plants by a variety of abiotic stresses (e.g., drought, low temperature), including metal stress [e.g., Cadmium (Cd), ammonium, Cupper (Cu) or Zink (Zn)], following treatment with ABA (Clement., et al., 2008, Gene. 426:15-22) or Ethylene, nutrient deprivation, and during senescence (Gepstein et al., 2003, Supra). In addition, tobacco plants transformed with the MT gene exhibit enhanced tolerance to low temperature, drought and salt stress (Xue T., 2009, J. Exp. Bot. 60:339-49).
Additional background art includes Beinsberger S E I, et al., 1992 [Effects of enhanced cytokinin levels in ipt transgenic tobacco. In: Kamínek M, Mok D W S, Zazímalová. E, editors. Physiology and Biochemistry of Cytokinins in Plants. The Hague, The Netherlands: SPB Academic Publishing; pp. 77-82].